Methods for generating hypermutable microbes

ABSTRACT

Bacteria are manipulated to create desirable output traits using dominant negative alleles of mismatch repair proteins. Enhanced hypermutation is achieved by combination of mismatch repair deficiency and exogenously applied mutagens. Stable bacteria containing desirable output traits are obtained by restoring mismatch repair activity to the bacteria.

This application claims the benefit of provisional application Ser. No.60/181,929 filed Feb. 11, 2000.

FIELD OF THE INVENTION

The invention is related to the area of mismatch repair genes. Inparticular it is related to the field of in situ mutagenesis of singlecelled organisms.

BACKGROUND OF THE INVENTION

Within the past four years, the genetic cause of the HereditaryNonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynchsyndrome II, has been ascertained for the majority of kindred's affectedwith the disease (14). The molecular basis of HNPCC involves geneticinstability resulting from defective mismatch repair (MMR). Severalgenes have been identified in humans that encode for proteins and appearto participate in the MMR process, including the mutS homologs GTBP,hMSH2, and hMSH3 and the mutL homologs hMLH1, hMLH3, hPMS1, and hPMS2(4,9,11,17,19,22,24,38). Germline mutations in four of these genes(hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindred's(13). Though the mutator defect that arises from the MMR deficiency canaffect any DNA sequence, microsatellite sequences are particularlysensitive to MMR abnormalities (14, 25, 27, 29). Microsatelliteinstability (MI) is therefore a useful indicator of defective MMR. Inaddition to its occurrence in virtually all tumors arising in HNPCCpatients, MI is found in a small fraction of sporadic tumors withdistinctive molecular and phenotypic properties (13).

HNPCC is inherited in an autosomal dominant fashion, so that the normalcells of affected family members contain one mutant allele of therelevant MMR gene (inherited from an affected parent) and one wild-typeallele (inherited from the unaffected parent). During the early stagesof tumor development, however, the wild-type allele is inactivatedthrough a somatic mutation, leaving the cell with no functional MMR geneand resulting in a profound defect in MMR activity. Because a somaticmutation in addition to a germ-line mutation is required to generatedefective MMR in the tumor cells, this mechanism is generally referredto as one involving two hits, analogous to the biallelic inactivation oftumor suppressor genes that initiate other hereditary cancers. In linewith this two-hit mechanism, the non-neoplastic cells of HNPCC patientsgenerally retain near normal levels of MMR activity due to the presenceof the wild-type allele (11, 13, 24). In addition, similar findings areobserved in other diploid organisms (2, 5, 8).

The ability to alter signal transduction pathways by manipulation of agene product's function, either by over-expression of the wild typeprotein or a fragment thereof, or by introduction of mutations intospecific protein domains of the protein, the so-called dominant-negativeinhibitory mutant, were described over a decade ago in the yeast systemSaccharomyces cerevisiae by Herskowitz (Nature 329:219–222, 1987). Ithas been demonstrated that over-expression of wild type gene productscan result in a similar, dominant-negative inhibitory phenotype due mostlikely to the “saturating-out” of a factor, such as a protein, that ispresent at low levels and necessary for activity; removal of the proteinby binding to a high level of its cognate partner results in the samenet effect, leading to inactivation of the protein and the associatedsignal transduction pathway.

Recently, work done by Nicolaides et.al. (32) has demonstrated theutility of introducing dominant negative inhibitory mismatch repairmutants into mammalian cells to confer global DNA hypermutability. Thereis a need in the art for additional techniques for generating mutationsin bacteria which can be used to make strains for production,biocatalysis, bioremediation, and drug discovery.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forrendering bacterial cells hypermutable.

It is another object of the present invention to provide geneticallyaltered bacteria.

It is yet another object of the present invention to provide a method toproduce bacteria that are hypermutable.

It is an object of the invention to provide a method to inactivate theprocess that results in hypermutable cells following strain selection.

It is a further object of the invention to provide a method of mutatinga gene of interest in a bacterium.

These and other embodiments of the invention are provided by one or moreof the embodiments described below. In one embodiment, a method isprovided for making a hypermutable bacteria. A polynucleotide comprisinga dominant negative allele of a mismatch repair gene is introduced intoa bacterium, whereby the cell becomes hypermutable. Preferably theallele is under the control of an inducible transcription regulatorysequence.

According to another aspect of the invention a homogeneous compositionof cultured, hypermutable, bacteria is provided. The bacteria comprise adominant negative allele of a mismatch repair gene. Preferably theallele is under the control of an inducible transcription regulatorysequence.

Another embodiment of the invention provides a method for generating amutation in a gene of interest. A bacterial culture comprising the geneof interest and a dominant negative allele of a mismatch repair gene isgrown. The cell is hypermutable. It is tested to determine whether thegene of interest harbors a mutation. Preferably the allele is under thecontrol of an inducible transcription regulatory sequence.

According to still another aspect of the invention a method forgenerating a mutation in a gene of interest is provided. A bacteriumcomprising the gene of interest and a dominant negative allele of amismatch repair gene is grown to form a population of mutated bacteria.The population of mutated bacteria is cultivated under trait selectionconditions. At least one of the cultivated bacteria is tested todetermine that the gene of interest harbors a mutation. Preferably theallele is under the control of an inducible transcription regulatorysequence.

Still another aspect of the invention is a method for enhancing themutation rate of a bacterium. A bacterium comprising a dominant negativeallele of an MMR gene is exposed to a mutagen whereby the mutation rateof the bacterium is enhanced in excess of the rate in the absence ofmutagen and in excess of the rate in the absence of the dominantnegative allele. Preferably the allele is under the control of aninducible transcription regulatory sequence.

Yet another aspect of the invention is a method for generating anMMR-proficient bacterium with a new output trait. A mismatch repairdeficient bacterium comprising a gene of interest and a dominantnegative allele of a mismatch repair gene is grown to form a populationof mutated bacteria. The population of mutated bacteria is cultivatedunder trait selection conditions. At least one of the cultivatedbacteria is tested to determine that the gene of interest harbors amutation. Mismatch repair activity is restored to the at least one ofthe cultivated bacteria. Preferably the allele is under the control ofan inducible transcription regulatory sequence.

These and other embodiments of the invention provide the art withmethods that can generate enhanced mutability in bacteria as well asproviding prokaryotic organisms harboring potentially useful mutationsto generate novel output traits for commercial applications. The abilityto create hypermutable organisms using dominant negative alleles hasgreat commercial value for the generation of innovative bacterialstrains that display new output features useful for a variety ofapplications, including but not limited to the manufacturing industryfor the generation of new biochemicals useful for detoxifying noxiouschemicals from by-products of manufacturing processes or those used ascatalysts, as well as helping in remediation of toxins present in theenvironment, including but not limited to polychlorobenzenes (PCBs),heavy metals and other environmental hazards for which there is a needto remove them from the environment. In addition to obtaining organismsthat are useful for removal of toxins from the environment, novelmicrobes can be selected for enhanced activity to either produceincreased quantity or quality of a protein or non-protein therapeuticmolecule by means of biotransformation (3). Biotransformation is theenzymatic conversion, by a microbe or an extract derived from themicrobe, of one chemical intermediate to the next product. There aremany examples of biotransformation in use for the commercialmanufacturing of important biological and chemical products, includingPenicillin G, Erythromycin, and Clavulanic Acid as well as organismsthat are efficient at conversion of “raw” materials to advancedintermediates and/or final products (Berry, A. Trends Biotechnol.14(7):250–256). The ability to control DNA hypermutability in hostbacterial strains using a dominant negative MMR (as described above)allows for the generation of variant subtypes that can be selected fornew phenotypes of commercial interest, including but not limited toorganisms that are toxin-resistant, have the capacity to degrade a toxinin situ or the ability to convert a molecule from an intermediate toeither an advanced intermediate or a final product. Other applicationsusing dominant negative MMR genes to produce genetic alteration ofbacterial hosts for new output traits include but are not limited torecombinant production strains that produce higher quantities of arecombinant polypeptide as well as the use of altered endogenous genesthat can transform chemical or catalyze manufacturing downstreamprocesses.

This application teaches of the use of a regulatable dominant negativeMMR phenotype to produce a prokaryotic strain with a commerciallybeneficial output trait. Using this process, microbes expressing adominant negative MMR can be directly selected for the phenotype ofinterest. Once a selected bacterium with a specified output trait isisolated, the hypermutable activity of the dominant negative MMR allelecan be turned-off by several methods well known to those skilled in theart. For example, if the dominant-negative allele is expressed by aninducible promoter system, including but not limited to promoters suchas: TAC-LACI, tryp (Brosius et.al. Gene 27:161–172, 1984), araBAD(Guzman et.al., J. Bact. 177:4121–4130, 1995) pLex (La Vallie et.al.,Bio.Technology 11:187–193, 1992), pRSET (Schoepfer, R. Gene 124:83–85,1993) , pT7 (Studier J. Mol. Biol. 219(1):37–44, 1991) etc., the induceris removed and the promoter activity is reduced, or a system thatexcises the MMR gene insert from the host cells harboring the expressionvector such as the Cre-lox (Hasan, N. et. al. Gene 2:51–56, 1994), aswell as methods that can homologously knockout of the expression vector.In addition to the recombinant methods outlined above that have thecapacity to eliminate the MMR activity from the microbe, it has beendemonstrated that many chemicals have the ability to “cure” microbialcells of plasmids. For example, chemical treatment of cells with drugsincluding bleomycin (Attfield et al. Antimicrob. Agents Chemother.27:985–988, 1985) or novobiocin, coumermycin, and quinolones (Fu et al.Chemotherapy 34:415–418, 1988) have been shown to result in microbialcells that lack endogenous plasmid as evidenced by Southern analysis ofcured cells as well as sensitivity to the appropriate antibiotic (1,41–43). Whether by use of recombinant means or treatment of cells withchemicals, removal of the MMR-expression plasmid results in there-establishment of a genetically stable microbial cell-line. Therefore,the restoration of MMR allows host bacteria to function normally torepair DNA. The newly generated mutant bacterial strain that exhibits anovel, selected output trait is now suitable for a wide range ofcommercial processes or for gene/protein discovery to identify newbiomolecules that are involved in generating a particular output trait.

While it has been documented that MMR deficiency can lead to as much asa 1000-fold increase in the endogenous DNA mutation rate of a host,there is no assurance that MMR deficiency alone will be sufficient toalter every gene within the DNA of the host bacterium to create alteredbiochemicals with new activity(s). Therefore, the use of chemical agentsand their respective analogues such as ethidium bromide, EMS, MNNG, MNU,Tamoxifen, 8-Hydroxyguanine, as well as others listed but not limited toin publications by: Khromov-Borisov, N. N., et. al. (Mutat. Res.430:55–74, 1999); Ohe, T., et. al. (Mutat. Res. 429:189–199, 1999);Hour, T. C. et. al. (Food Chem. Toxicol. 37:569–579, 1999); Hrelia, P.,et. al. (Chem. Biol. Interact. 118:99–111, 1999); Garganta, F., et. al.(Environ. Mol. Mutagen. 33:75–85, 1999); Ukawa-Ishikawa S., et. al.(Mutat. Res. 412:99–107, 1998); the website having the URL address: wwwhost server, ehs.utah.edu domain name, ohh directory, mutagenssubdirectory, etc. can be used to further enhance the spectrum ofmutations and increase the likelihood of obtaining alterations in one ormore genes that can in turn generate host bacteria with a desired newoutput trait(s) (10,39,40). Prior art teaches that mismatch repairdeficiency leads to hosts with an increased resistance to toxicity bychemicals with DNA damaging activity. This feature allows for thecreation of additional genetically diverse hosts when mismatch defectivebacteria are exposed to such agents, which would be otherwise impossibledue to the toxic effects of such chemical mutagens [Colella, G., et. al.(Br. J. Cancer 80:338–343, 1999); Moreland, N. J., et. al. (Cancer Res.59:2102–2106, 1999); Humbert, O., et. al. (Carcinogenesis 20:205–214,1999); Glaab, W. E., et. al. (Mutat. Res. 398:197–207, 1998)]. Moreover,prior art teaches that mismatch repair is responsible for repairingchemical-induced DNA adducts, so therefore blocking this process couldtheoretically increase the number, types, mutation rate and genomicalterations of a bacterial host [Rasmussen, L. J. et. al.(Carcinogenesis 17:2085–2088, 1996); Sledziewska-Gojska, E., et. al.(Mutat. Res. 383:31–37, 1997); and Janion, C. et. al. (Mutat. Res.210:15–22, 1989)]. In addition to the chemicals listed above, othertypes of DNA mutagens include ionizing radiation and UV-irradiation,which are known to cause DNA mutagenesis in bacteria can also be used topotentially enhance this process. These agents which are extremely toxicto host cells and therefore result in a decrease in the actual pool sizeof altered bacterial cells are more tolerated in MMR defective hosts andin turn allow for a enriched spectrum and degree of genomic mutation(7).

This application teaches new uses of MMR deficient bacterial cells tocreate commercially viable microbes that express novel output traits.Moreover, this application teaches the use of dominant negative MMRgenes to decrease the endogenous MMR activity of the host followed byplacing the cells under selection to obtain a desired, sought afteroutput trait for commercial applications such as but not limited torecombinant manufacturing, biotransformation and bioremediation.Furthermore, the application teaches the use of restoring MMR activityto the hypermutable bacterial host following strain selection of thevariant of interest as a means to genetically “fix” the new mutations inthe host genome. The application also teaches the use of enhancedhypermutability in bacteria by using MMR deficiency and chemical orradiation mutagenesis to create variant subtypes of bacteria useful forcommercial and other applications. The application describes uses ofhypermutable bacteria for producing strains that can be used to generatenew output traits for chemical manufacturing, pharmaceutical and othercommercially applicable processes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Western blot of steady-state PMS134 levels in IPTG-treatedsamples in DH10B strain. Cells containing the pTACPMS134 (lane 2) showeda robust steady state level of protein after induction in contrast tocells expressing empty vector (lane 1). Blots were probed with ananti-human-PMS2 polyclonal antibody.

FIG. 2. Western blot of PMS134V5 and PMSR3V5 in IPTG-treated (+) anduntreated (−) samples in BL21 strain. Blots were probed with an anti-V5antibody, which is directed to the C-terminal tag of each protein.

FIG. 3. Number of Kanamycin resistant PMS134 and vector control DH10Bclones. IPTG-induced strains were grown and plated onto KAN plates andgrown for 18 hours at 37° C. to identify number of KAN resistant clonesdue to genetic alteration.

FIG. 4. Number of Kanamycin Resistant PMS134, PMSR3 and vector controlBL21 clones. IPTG-induced strains were grown and plated onto AMP and KANplates and grown for 18 hours at 37° C. to identify number of KANresistant clones due to genetic alteration.

FIG. 5. (A) Western blot of steady-state ATPMS134flag in IPTG-treatedsamples in DH10B. Lysates from untransfected cells (lane 1) and abacterial clone expressing the Arabidopsis thaliana PMS134 truncatedprotein with a FLAG epitope fused to the C-terminus (ATPMS134flag) (lane2) were electrophoresed on SDS-PAGE gels. Blots were probed with ananti-FLAG monoclonal antibody directed to the FLAG epitope. (B) Numberof Kanamycin Resistant ATPMS134flag and vector control DH10B clones.IPTG-induced strains were grown and plated onto AMP and KAN plates andgrown for an additional 18 hours at 37° C. to identify number of KANresistant clones due to genetic alteration.

FIG. 6. Generation of high recombinant producer BGAL-MOR lines in PMS134expressing DH5alpha host strains.

DETAILED DESCRIPTION OF THE INVENTION

The inventors present a method for developing hypermutable bacteria byaltering the activity of endogenous mismatch repair activity of hosts.Wild type and some dominant negative alleles of mismatch repair genes,when introduced and expressed in bacteria, increase the rate ofspontaneous mutations by reducing the effectiveness of the endogenousMMR-mediated DNA repair activity, thereby rendering the bacteria highlysusceptible to genetic alterations due to hypermutability. Hypermutablebacteria can then be utilized to screen for novel mutations in a gene ora set of genes that produce variant siblings that exhibit a new outputtrait(s) not found in the wild type cells.

The process of mismatch repair, also called mismatch proofreading, is anevolutionarily highly conserved process that is carried out by proteincomplexes described in cells as disparate as prokaryotic cells such asbacteria to more complex mammalian cells (14, 29, 31, 33, 34). Amismatch repair gene is a gene that encodes one of the proteins of sucha mismatch repair complex. Although not wanting to be bound by anyparticular theory of mechanism of action, a mismatch repair complex isbelieved to detect distortions of the DNA helix resulting fromnon-complementary pairing of nucleotide bases. The non-complementarybase on the newer DNA strand is excised, and the excised base isreplaced with the appropriate base that is complementary to the olderDNA strand. In this way, cells eliminate many mutations that occur as aresult of mistakes in DNA replication, resulting in genetic stability ofthe sibling cells derived from the parental cell.

Some wild type alleles as well as dominant negative alleles cause amismatch repair defective phenotype even in the presence of a wild-typeallele in the same cell. An example of a dominant negative allele of amismatch repair gene is the human gene hPMS2-134, which carries atruncation mutation at codon 134 (32). The mutation causes the productof this gene to abnormally terminate at the position of the 134th aminoacid, resulting in a shortened polypeptide containing the N-terminal 133amino acids. Such a mutation causes an increase in the rate ofmutations, which accumulate in cells after DNA replication. Expressionof a dominant negative allele of a mismatch repair gene results inimpairment of mismatch repair activity, even in the presence of thewild-type allele. Any mismatch repair allele, which produces sucheffect, can be used in this invention. In addition, the use ofover-expressed wildtype MMR gene alleles from human, mouse, plants, andyeast in bacteria has been shown to cause a dominant negative effect onthe bacterial hosts MMR activity (9, 33, 34, 38).

Dominant negative alleles of a mismatch repair gene can be obtained fromthe cells of humans, animals, yeast, bacteria, plants or otherorganisms. Screening cells for defective mismatch repair activity canidentify such alleles. Mismatch repair genes may be mutant or wild type.Bacterial host MMR may be mutated or not. The term bacteria used in thisapplication include any organism from the prokaryotic kingdom. Theseorganisms include genera such as but not limited to Agrobacterium,Anaerobacter, Aquabacterium, Azorhizobium, Bacillus, Bradyrhizobium,Cryobacterium, Escherichia, Enterococcus, Heliobacterium, Klebsiella,Lactobacillus, Methanococcus, Methanothermobacter, Micrococcus,Mycobacterium, Oceanomonas, Pseudomonas, Rhizobium, Staphylococcus,Streptococcus, Streptomyces, Thermusaquaticus, Thermaerobacter,Thermobacillus, etc. Other procaryotes that can be used for thisapplication are listed at the website having the URL address www.hostserver, bacterio.cict.fr domain name, validgenericnames directory.Bacteria exposed to chemical mutagens or radiation exposure can bescreened for defective mismatch repair. Genomic DNA, cDNA, or mRNA fromany cell encoding a mismatch repair protein can be analyzed forvariations from the wild type sequence. Dominant negative alleles of amismatch repair gene can also be created artificially, for example, byproducing variants of the hPMS2-134 allele or other mismatch repairgenes (32). Various techniques of site-directed mutagenesis can be used.The suitability of such alleles, whether natural or artificial, for usein generating hypermutable bacteria can be evaluated by testing themismatch repair activity (using methods described in ref 32) caused bythe allele in the presence of one or more wild-type alleles, todetermine if it is a dominant negative allele.

A bacterium that over-expresses a wild type mismatch repair allele as ora dominant negative allele of a mismatch repair gene will becomehypermutable. This means that the spontaneous mutation rate of suchbacteria is elevated compared to bacteria without such alleles. Thedegree of elevation of the spontaneous mutation rate can be at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,or 1000-fold that of the normal bacteria as measured as a function ofbacterial doubling/minute.

According to one aspect of the invention, a polynucleotide encodingeither a wild type or a dominant negative form of a mismatch repairprotein is introduced into bacteria. The gene can be any dominantnegative allele encoding a protein which is part of a mismatch repaircomplex, for example, mutS, mutL, mutH, or mutY homologs of thebacterial, yeast, plant or mammalian genes (14, 28). The dominantnegative allele can be naturally occurring or made in the laboratory.The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or achemically synthesized polynucleotide or polypeptide. The molecule canbe introduced into the cell by transfection, transformation,conjugation, fusion, or other methods well described in the literature.

Any process can be used whereby a polynucleotide or polypeptide isintroduced into a cell. The process of gene transfer can be carried outin a bacterial culture using a suspension culture. The bacteria can beany type classified under the prokaryotes.

In general, gene transfer will be carried out using a suspension ofcells but other methods can also be employed as long as a sufficientfraction of the treated cells incorporate the polynucleotide orpolypeptide so as to allow recipient cells to be grown and utilized. Theprotein product of the polynucleotide may be transiently or stablyexpressed in the cell. Techniques for gene transfer are well known tothose skilled in the art. Available techniques to introduce apolynucleotide or polypeptide into a prokaryote include but are notlimited to electroporation, transduction, cell fusion, the use ofchemically competent cells (e.g. calcium chloride), and packaging of thepolynucleotide together with lipid for fusion with the cells ofinterest. Once a cell has been transformed with the dominant negativemismatch repair gene or protein, the cell can be propagated andmanipulated in either liquid culture or on a solid agar matrix, such asa petri dish. If the transfected cell is stable, the gene will beretained and expressed at a consistent level when the promoter isconstitutively active, or when in the presence of appropriate inducermolecules when the promoter is inducible, for many cell generations, anda stable, hypermutable bacterial strain results.

An isolated bacterial cell is a clone obtained from a pool of abacterial culture by chemically selecting out non-recipient strainsusing, for example, antibiotic selection of an expression vector. If thebacterial cell is derived from a single cell, it is defined as a clone.

A polynucleotide encoding a dominant negative form of a mismatch repairprotein can be introduced into the genome of a bacterium or propagatedon an extra-chromosomal plasmid. Selection of clones harboring themismatch repair gene expression vector can be accomplished by additionof any of several different antibiotics, including but not limited toampicillin, kanamycin, chloramphenicol, zeocin, and tetracycline. Themicrobe can be any species for which suitable techniques are availableto produce transgenic microorganisms, such as but not limited to generaincluding Bacillus, Pseudomonas, Staphylococcus, Escherichia and others.

Any method for making transgenic bacteria known in the art can be used.According to one process of producing a transgenic microorganism, thepolynucleotide is transfected into the microbe by one of the methodswell known to those in the art. Next, the microbial culture is grownunder conditions that select for cells in which the polynucleotideencoding the mismatch repair gene is either incorporated into the hostgenome as a stable entity or propagated on a self-replicatingextra-chromosomal plasmid, and the protein encoded by the polynucleotidefragment transcribed and subsequently translated into a functionalprotein within the cell. Once transgenic microbe is engineered to harborthe expression construct, it is then propagated to generate and sustaina culture of transgenic microbes indefinitely.

Once a stable, transgenic microorganism has been engineered to express afunctional mismatch repair (MMR) protein, the microbe can be exploitedto create novel mutations in one or more target gene(s) of interestharbored within the same microorganism. A gene of interest can be anygene naturally possessed by the bacterium or one introduced into thebacterial host by standard recombinant DNA techniques. The targetgene(s) may be known prior to the selection or unknown. One advantage ofemploying such transgenic microbes to induce mutations in resident orextra-chromosomal genes within the microbe is that it is unnecessary toexpose the microorganism to mutagenic insult, whether it be chemical orradiation in nature, to produce a series of random gene alterations inthe target gene(s). This is due to the highly efficient nature and thespectrum of naturally occurring mutations that result as a consequenceof the altered mismatch repair process. However, it is possible toincrease the spectrum and frequency of mutations by the concomitant useof either chemicals and/or radiation together with MMR defective cells.These include DNA mutagens, DNA alkylating agents, DNA intercalatingagents, DNA oxidizing agents, ionizing radiation, and ultravioletradiation. The net effect of the combination treatment is the increasein altered gene pool in the genetically altered microbe that result inan increased alteration of an allele(s) that are useful for producingnew output traits. Another benefit of using MMR-defective microbes thatare taught in this application is that one can perform a genetic screenfor the direct selection of variant sub-clones that exhibit new outputtraits with commercially important applications. This allows one tobypass tedious and time consuming gene identification, isolation andcharacterization.

Mutations can be detected by analyzing the recombinant microbe foralterations in the genotype and/or phenotype post-activation of thedecreased mismatch repair activity of the transgenic microorganism.Novel genes that produce altered phenotypes in MMR-defective microbialcells can be discerned by any variety of molecular techniques well knownto those in the art. For example, the microbial genome can be isolatedand a library of restriction fragments cloned into a plasmid vector. Thelibrary can be introduced into a “normal” cell and the cells exhibitingthe novel phenotype screened. A plasmid is isolated from those normalcells that exhibit the novel phenotype and the gene(s) characterized byDNA sequence analysis. Alternatively, differential messenger RNA screencan be employed utilizing driver and tester RNA (derived from wild typeand novel mutant respectively) followed by cloning the differentialtranscripts and characterizing them by standard molecular biologymethods well known to those skilled in the art. Furthermore, if themutant sought is on encoded by an extrachromosomal plasmid, thenfollowing co-expression of the dominant negative MMR gene and the geneof interest to be altered and phenotypic selection, the plasmid isisolated from mutant clones and analyzed by DNA sequence analysis bymethods well known to those in the art. Phenotypic screening for outputtraits in MMR-defective mutants can be by biochemical activity and/or aphysical phenotype of the altered gene product. A mutant phenotype canalso be detected by identifying alterations in electrophoretic mobility,DNA binding in the case of transcription factors, spectroscopicproperties such as IR, CD, X-ray crystallography or high field NMRanalysis, or other physical or structural characteristics of a proteinencoded by a mutant gene. It is also possible to screen for alterednovel function of a protein in situ, in isolated form, or in modelsystems. One can screen for alteration of any property of themicroorganism associated with the function of the gene of interest,whether the gene is known prior to the selection or unknown. Theaforementioned screening and selection discussion is meant to illustratethe potential means of obtaining novel mutants with commerciallyvaluable output traits.

Plasmid expression vectors that harbor the mismatch repair (MMR) geneinserts can be used in combination with a number of commerciallyavailable regulatory sequences to control both the temporal andquantitative biochemical expression level of the dominant negative MMRprotein. The regulatory sequences can be comprised of a promoter,enhancer or promoter/enhancer combination and can be inserted eitherupstream or downstream of the MMR gene to control the expression level.The regulatory promoter sequence can be any of those well known to thosein the art, including but not limited to the lac, tetracycline,tryptophan-inducible, phosphate inducible, T7-polymerase-inducible (30),and steroid inducible constructs as well as sequences which can resultin the excision of the dominant negative mismatch repair gene such asthose of the Cre-Lox system. These types of regulatory systems arefamiliar to those skilled in the art.

Once a microorganism with a novel, desired output trait of interest iscreated, the activity of the aberrant MMR activity can be attenuated oreliminated by any of a variety of methods, including removal of theinducer from the culture medium that is responsible for promoteractivation, gene disruption of the aberrant MMR gene constructs,electroporation and/or chemical curing of the expression plasmids(Brosius, Biotechnology 10:205–225,1988; Wang et. al., J. of FujianAgricultural University 28:43–46,1999; Fu et. al., Chem Abstracts34:415–418, 1988). The resulting microbe is now useful as a stablestrain that can be applied to various commercial applications, dependingupon the selection process placed upon it.

In cases where genetically deficient mismatch repair bacteria [strainssuch as but not limited to: M1 (mutS) and in EC2416 (mutS delta umuDC),and mutL or mutY strains] are used to derive new output traits,transgenic constructs can be used that express wild-type mismatch repairgenes sufficient to complement the genetic defect and therefore restoremismatch repair activity of the host after trait selection [Grzesiuk, E.et. al. (Mutagenesis 13:127–132, 1998); Bridges, B. A., et. al. (EMBO J.16:3349–3356, 1997); LeClerc, J. E., Science 15:1208–1211, 1996);Jaworski, A. et. al. (Proc. Natl. Acad. Sci USA 92:11019–11023, 1995)].The resulting microbe is genetically stable and can be applied tovarious commercial practices. The use of over expressing foreignmismatch repair genes from human and yeast such as PMS1, MSH2, MLH1,MLH3, etc. have been previously demonstrated to produce a dominantnegative mutator phenotype in bacterial hosts (35, 36, 37). In addition,the use of bacterial strains expressing prokaryotic dominant negativeMMR genes as well as hosts that have genomic defects in endogenous MMRproteins have also been previously shown to result in a dominantnegative mutator phenotype (29,32). However, the findings disclosed hereteach the use of MMR genes, including the human PMSR2 and PMSR3 gene(ref 19), the related PMS134 truncated MMR gene (ref 32), the plantmismatch repair genes and those genes that are homologous to the 134N-terminal amino acids of the PMS2 gene which include the MutL family ofMMR proteins and including the PMSR and PMS2L homologs described by Horiet. al. (accession number NM₁₃ 005394 and NM₁₃ 005395) and Nicolaides(reference 19) to create hypermutable microbes. In addition, thisapplication teaches the use of DNA mutagens in combination with MMRdefective microbial hosts to enhance the hypermutable production ofgenetic alterations. This accentuates MMR activity for generation ofmicroorganisms with commercially relevant output traits such as but notlimited to recombinant protein production strains, biotransformation,and bioremediation.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples that will be provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention

EXAMPLES Example 1 Generation of Inducible MMR Dominant Negative AlleleVectors

Bacterial expression constructs were prepared to determine if the humanPMS2 related gene (hPMSR3) (19) and the human PMS134 gene (32) arecapable of inactivating the bacterial MMR activity and thereby increasethe overall frequency of genomic hypermutation, a consequence of whichis the generation of variant sib cells with novel output traitsfollowing host selection. Moreover, the use of regulatable expressionvectors will allow for suppression of dominant negative MMR alleles andrestoration of the MMR pathway and genetic stability in hosts cells(43). For these studies, a plasmid encoding the hPMS134 cDNA was alteredby polymerase chain reaction (PCR). The 5′ oligonucleotide has thefollowing structure: 5′-ACG CAT ATG GAG CGA GCT GAG AGC TCG AGT-3′ (SEQID NO: 1) that includes the NdeI restriction site CAT ATG. The3′-oligonucleotide has the following structure: 5′-GAA TTC TTA TCA CGTAGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC AGT TCC AAC CTT CGCCGA TGC-3′ (SEQ ID NO: 2) that includes an EcoRI site GAA TTC and the 14amino acid epitope for the V5 antibody. The oligonucleotides were usedfor PCR under standard conditions that included 25 cycles of PCR (95° C.for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25 cyclesfollowed by 3 minutes at 72° C). The PCR fragment was purified by gelelectrophoresis and cloned into pTA2.1 (InVitrogen) by standard cloningmethods (Sambrook et al., Molecular Cloning: A Laboratory Manual, ThirdEdition, 2001), creating the plasmid pTA2.1-hPMS134. pTA2.1-hPMS134 wasdigested with the restriction enzyme EcoRI to release the insert (thereare two EcoRI restriction sites in the multiple cloning site of pTA2.1that flank the insert) and the fragment filled in with Klenow fragmentand dNTPs. Next, the fragment was gel purified, then digested with NdeIand inserted in pT7-Ea that had been digested with NdeI and BamHI(filled with Klenow) and phosphatase treated. The new plasmid wasdesignated pT7-Ea-hPMS134. The following strategy, similar to thatdescribed above to clone human PMS134, was used to construct anexpression vector for the human related gene PMSR3. First, the hPMSR3fragment was amplified by PCR to introduce two restriction sites, anNdeI restriction site at the 5′-end and an Eco RI site at the 3′-end ofthe fragment. The 5′-oligonucleotide that was used for PCR has thefollowing structure: 5′-ACG CAT ATG TGT CCT TGG CGG CCT AGA-3′ (SEQ IDNO: 3) that includes the NdeI restriction site CAT ATG. The3′-oligonucleotide used for PCR has the following structure: 5′-GAA TTCTTA TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC CAT GTGTGA TGT TTC AGA GCT-3′ (SEQ ID NO: 4) that includes an EcoRI site GAATTC and the V5 epitope to allow for antibody detection. The plasmid thatcontained human PMSR3 in pBluescript SK (19) was used as the PCR targetwith the hPMS2-specific oligonucleotides above. Following 25 cycles ofPCR (95° C. for 1 minute, 55° C for 1 minute, 72° C. for 1.5 minutes for25 cycles followed by 3 minutes at 72° C.). The PCR fragment waspurified by gel electrophoresis and cloned into pTA2.1 (InVitrogen) bystandard cloning methods (Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, 2001), creating the plasmidpTA2.1-hR3. pTA2.1-hR3 was next digested with the restriction enzymeEcoRI to release the insert (there are two EcoRI restriction sites inthe multiple cloning site of pTA2.1 that flank the insert) and thefragment filled in with Klenow fragment and dNTPs. Then, the fragmentwas gel purified, then digested with NdeI and inserted in pT7-Ea thathad been digested with NdeI and BamHI (filled with Klenow) andphosphatase treated. The new plasmid was designated pT7-Ea-hR3.

BL21 cells harbor an additional expression vector for the lysozymeprotein, which has been demonstrated to bind to the T7 polymerase insitu; this results in a bacterial strain that has very low levels of T7polymerase expression. However, upon addition of the inducer IPTG, thecells express high-levels of T7 polymerase due to the IPTG-inducibleelement that drives expression of the polymerase that is resident withinthe genome of the BL21 cells (30). The BL21 cells are chloramphenicolresistant due to the plasmid that expresses lysozyme within the cell. Tointroduce the pT7-hPMS134 or the pT7-hPMSR3 genes into BL21 cells, thecells were made competent by incubating the cells in ice cold 50mM CaCl₂for 20 minutes, followed by concentrating the cells and addingsuper-coiled plasmid DNA as describe (Maniatis, T. et. al. Cold SpringHarbor Laboratory Press, Third Edition, 2001). Ampicillin resistant BL21were selected on LB-agar plates [5% yeast extract, 10% bactotryptone, 5%NaCl, 1.5% bactoagar, pH 7.0 (Difco)] plates containing 25 μg/mlchloramphenicol and 100 μg/ml ampicillin. The next day, bacterialcolonies were picked and analyzed for vectors containing an intactpTACPMS134 or pTAC empty vector by restriction endonuclease digestionand sequence analysis.

In addition to constructing a V5-epitope tagged PMS134 construct we alsoconstructed and tested a non-epitope tagged version. This was preparedto demonstrate that the simple fact of epitope tagging the construct didnot result in alteration of the dominant-negative phenotype that PMS134has on mismatch repair activity. For these studies, a BamHI restrictionfragment containing the hPMS134 cDNA was filled-in with Klenow fragmentand then sub-cloned into a Klenow-filled blunt-ended NdeI-XhoI site ofthe pTACLAC expression vector, which contains theisopropylthio-β-galactosidase (IPTG)-inducible bacterial TAC promoterand ampicillin resistance gene as selectable marker. The NdeI-XhoIcloning site is flanked by the TACLAC promoter that contains the LacIrepressor site followed by a Shine Dalgarno ribosome-binding site at the5′ flanking region and the T1 T2 ribosomal RNA terminator in the 3′flanking region. The TACLAC vector also contains the LacI gene, which isconstitutively expressed by the TAC promoter.

DH10B bacterial cells containing the pBCSK vector (Stratagene), whichconstitutively expresses the β-galactosidase gene and contains thechloramphenicol resistance marker for selection, were made competent viathe CaCl₂ method (Maniatis, T. et. al. Cold Spring Harbor LaboratoryPress, 1982). This vector turns bacterial cells blue when grown in thepresence of IPTG and X-gal that aids in the detection of bacterialcolonies. Competent cells were transfected with the pTAC empty vector orthe pTACPMS134 vector following the heat-shock protocol. Transfectedcultures were plated onto LB-agar [5% yeast extract, 10% bactotryptone,5% NaCl, 1.5% bactoagar, pH 7.0 (Difco)] plates containing 25 μg/mlchloramphenicol and 100 μ/ml ampicillin. The next day, bacterialcolonies were picked and analyzed for vectors containing an intactpTACPMS134 or pTAC empty vector by restriction endonuclease digestionand sequence analysis. Ten clones of each bacteria containing correctempty or PMS134 inserts were then grown to confluence overnight in LBmedia (5% yeast extract, 10% bactotryptone, 5% NaCl, pH 7.0) containing10 μg/ml chloramphenicol and 50 μg/ml ampicillin. The next day TAC emptyor pTACPMS134 cultures were diluted 1:4 in LB medium plus 50 μM IPTG(Gold Biotechnology) and cultures were grown for 12 and 24 hours at 37°C. After incubation, 50 μl aliquots were taken from each culture andadded to 150 μls of 2×SDS buffer and cultures were analyzed for PMS134protein expression by western blot.

Western blots were carried out as follows. 50 μls of each PMS134 orempty vector culture was directly lysed in 2×lysis buffer (60 mM Tris,pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001%bromophenol blue) and samples were boiled for 5 minutes. Lysate proteinswere separated by electrophoresis on 4–20% Tris glycine gels (Novex).Gels were electroblotted onto Immobilon-P (Millipore) in 48 mM Trisbase, 40 mM glycine, 0.0375% SDS, 20% methanol and blocked overnight at4° C. in Tris-buffered saline plus 0.05% Tween-20 and 5% condensed milk.Filters were probed with a rabbit polyclonal antibody generated againstthe N-terminus of the human PMS2 polypeptide (Santa Cruz), which is ableto recognize the PMS134 polypeptide (31), followed by a secondary goatanti-rabbit horseradish peroxidase-conjugated antibody. After incubationwith the secondary antibody, blots are developed using chemiluminescence(Pierce) and exposed to film to measure PMS134 expression.

As shown in FIG. 1, a robust expression of PMS134 could be detected inbacterial cells containing pTACPMS134 (lane 2) in contrast to cellsexpressing empty vector (lane 1), which had no signal.

For induction of PMS134 and PMSR3 in BL21 cells, the pT7-Ea-hPMS134 orthe pT7-Ea-hPMSR3 cells were induced with 50 μM IPTG for 12 and 24hours. Cell lysates were prepared and analyzed by western blot listedabove using either the N-terminal PMS2 antibody to detect the PMS134containing cells or the antiV5-horseradish peroxidase conjugatedmonoclonal antibody (InVitrogen) to detect the PMS134V5 and PMSR3V5polypeptides. FIG. 2 shows the expression of PMS134V5 and PMSR3V5 before(−) lanes and after IPTG (+) lanes induction.

Example 2 Generation of Hypermutable Bacteria with Inducible DominantNegative Alleles of Mismatch Repair Genes

Bacterial clones expressing the PMS134 or the empty vector were grown inliquid culture for 24 hr at 37° C. in the presence of 10 μg/mlchloramphenicol and 50 μg/ml ampicillin plus 50 μM IPTG. The next day,cultures were diluted 1:10 in medium containing 50 μM IPTG plusampicillin/chloramphenicol (AC) or ampicillin/chloramphenicol plus 25μg/ml kanamycin (ACK) and cultures were grown for 18 hr at 37° C. Thefollowing day, a 0.1 μl aliquot (2 μl diluted in 1000 μl of LB mediumand used 50 μl for plating) of cells grown in AC medium were plated onLB-agar plates containing 40 μg/ml of5-bromo-4-chloro-3-indolyl-B-D-galactoside (X-gal) plus 100 μg/mlampicillin (AMP), while a 1 μl aliquot (1 μl diluted in 100 μl of LBmedium and used 100 μl for plating) of cells grown in ACK medium wereplated on LB-agar plates containing X-gal and 50 μg/ml kanamycin (KAN).Plates were incubated for 18 hours at 37° C. The results from thesestudies show that cells expressing the PMS134 were able to increasehypermutation in the genome of the DH10B bacterial strain which resultedin the production of siblings that exhibit new biological traits such asKAN resistance (FIG. 3).

Kanamycin-resistant assays using BL21 cells expressing the V5-tagged oruntagged PMS134 or PMSR3 polypeptides were carried out as describedabove. BL21 bacterial cells that harbor the empty vector, pT7-PMS134 orpT7-PMSR3 were grown overnight in LB supplemented with 100 μg/mlampicillin. The overnight cultures were diluted 1:100 into freshampicillin containing medium and grown for 2.5 hours at 37° C. withcontinuous shaking. When the cells reached an optical density (OD) of0.6, measured at 600 nm, IPTG was added to each culture to a finalconcentration of 0.5 mM. Cells were incubated for 24, and 48 hours; atthose time points cells were removed for SDS-PAGE analysis and plating(see above). BL21/pT7 (empty vector), BL21/pT7-PMS134, and BL21/pT7-R3cells were plated onto LB plates, LB plates that contained 100 μg/mlampicillin, and plates that contain 50 μg/ml Kanamycin. The equivalentof 1×10⁷ cells/plate were spread onto the plates. BL21 cells that harborthe empty vector are capable of growth on LB plates as well as LB platesthat contain 10 μg/ml ampicillin; that is as expected since the pT7expression vector renders the cells ampicillin resistant. The vectoronly control is not capable of growth on Kanamycin. After 24 hrIPTG-induction PMS134 or PMSR3 cells had a significant number of KANresistant cells while none were observed in BL21 parental cells grownunder similar conditions (FIG. 4). Moreover, BL21 cells containing thePMS134 or PMSR3 genes under non-IPTG-induced conditions failed toproduce any KAN resistant clones demonstrating the need for expressionof the PMS polypeptides for hypermutability. A summary outlining thedata and number of Kanamycin resistant bacterial clones is provided inTABLE 1.

TABLE 1 Generation of Kanamycin resistant clones via MMR deficiency #CELLS AMP^(R) KAN^(R) STRAIN SEEDED colonies colonies FREQUENCY DH10BVEC  50,000  62,000    0 0 DH10B  50,000  43,146   23 53 × 10^(″4)PMS134 BL21 VEC 500,000 520,800    0 0 BL21 T7-Ea- 500,000 450,000 2,2454.9 × 10⁻³ PMS134V5 BL21 T7-Ea- 500,000 500,000 1,535 3.1 × 10⁻³ PMSR3V5

These data demonstrate and enable the proof-of-concept that the use ofthe dominant negative MMR genes is a viable approach to creatinghypermutable bacteria that can lead to the generation of phenotypicallydiverse offspring when put under selective conditions.

Using the same protocol as listed above and the same cloning strategy, atruncated PMS2 homolog from the Arabidopsis thaliana plant, which wascloned by degenerate PCR from an Arabidopsis thaliana cDNA library(Strategene), was found to give a similar enhancement of genetichypermutability in DH5alpha bacteria FIG. 5. For detection purposes, wefused a FLAG epitope to the C-terminus of the PMS134 polypeptide usingPCR and an antisense primer directed to the 134 codon region of theArabidopsis PMS2 homolog followed by a FLAG epitope and 2 terminationcodons. The resultant fusion was termed ATPMS134-flag. The ATPMS134-flag gene was then cloned into the IPTG-inducible TACLACexpression vector and transfected into DH5alpha cells. Western blot ofbacteria transfected with an IPTG-inducible expression vector carrying atruncated version (codons 1–134) of the Arabidopsis thaliana PMS2homolog using the anti-FLAG antibody demonstrated the inducibility andsteady-state protein levels of the chimeric gene. FIG. 5A shows thewestern blot containing protein from an untransfected cell (lane 1) anda bacterial clone expressing the Arabidopsis PMS2-134 truncated protein(lane 2). Following the mutagenesis protocol described above, bacterialcells expressing the ATPMS134 protein were found to have an increase inthe number of KAN resistant cells (12 clones) in contrast to cellsexpressing the empty vector that yielded no KAN resistant clone.

Bacterial cells such as the pT7-PMS134 and pT7-R3 harboring BL21 cells;the TACLACPMS134 DH10B; the TACLACMLH1 DH10B cells; or theTACLAC-ATPMS134flag DH5alpha cells are capable of growth on LB,LB/ampicillin and LB/KAN plates because the cells have acquiredmutations within their genome that render the cell drug resistant. Cellsthat express dominant negative MMR genes have altered the mismatchcontrol pathway of the microbe, presumably altering a gene or a set ofgenes that control resistance to kanamycin. A new output trait,Kanamycin-resistance, is generated by expression of the dominantnegative MMR gene in these cells. These data demonstrate the ability ofdominant negative MMR genes to produce hypermutability across a widearray of bacterial strains to produce new output traits such asKanamycin resistance.

EXAMPLE 3 Dominant Negative MMR Genes Can Produce New Genetic Variantsand Commercially Viable Output Traits in Prokaryotic Organisms

The data presented in EXAMPLE 2 show the ability to generate geneticalterations and new phenotypes in bacterial strains expressing dominantnegative MMR genes. In this EXAMPLE we teach the utility of this methodto create prokaryotic strains with commercially relevant output traits.

Generation of Heat-resistant Producer Strains

One example of commercial utility is the generation of heat-resistantrecombinant protein producer strains. In the scalable process ofrecombinant manufacturing, large-scale fermentation of prokaryotesresults in the generation of heat, which leads to suboptimal growthconditions for the producer strain and thus resulting in lowerrecombinant protein yields. In order to circumvent this problem, weemployed the use of DH10B bacteria containing the inducible TACLACPMS134gene. Briefly, cells were grown in 5 ml LB shake flasks containingampicillin and IPTG-induced for 0, 24 and 48 hrs at 37C. Cultures wereharvested and then incubated at 100C. for 0, 1 or 10 minutes (times atwhich 100% of the wild-type strain perishes) and 100 μl aliquots(equivalent to 250,000 cells) were plated onto LB agar plates containingampicillin to identify heat resistant clones. Table 2 shows a typicalexperiment whereby cells containing the TACLACPMS134 gene generated asignificant number of heat-resistant clones after 48 hours of PMS134induction and hypermutation via MMR blockade. No or a few clones wereobserved in the uninduced or 24 hr induced conditions respectivelysuggesting the needs for multiple rounds of genetic mutation to producegenes that are capable of allowing bacteria to survive under harshconditions. Similar results were observed with other dominant negativemutants such as the PMSR2, PMSR3, and the human MLH1 proteins (notshown).

TABLE 2 Generation of heat-resistant clones via MMR deficiency HeatedHeated Heated Treatment 0 min 1 min 10 min TACLACVEC 250,000 +/− 7,500 0 0 0 hr IPTG TACLACPMS134 265,000 +/− 2,000  0 0 hr IPTG TACLACVEC274,000 +/− 12,000 1 +/− 0 0 24 hr IPTG TACLACPMS134 240,000 +/− 9,400 5 +/− 2 0 24 hr IPTG TACLACVEC 256,000 +/− 12,000 0 0 48 hr IPTGTACLACPMS134 252,000 +/− 14,000 65 +/− 8  3 +− 1 48 hr IPTGGeneration of High Recombinant Protein Producer Strains.

Next, we tested the ability of bacteria expressing dominant negative MMRgenes to produce subclones with enhanced recombinant protein production.In these experiments again we employed the DH10B cells containing theTACLACPMS134 inducible vector plus the pTLACZ vector, whichconstitutively expresses the β-galactosidase gene. Analysis ofindividual clones containing the TACLACPMS134 and pTLACZ vectortypically produces 10–20 μg/ml of LACZ protein via shake flaskfermentation after IPTG induction for 24 hours. To test the hypothesisthat high recombinant producer strains can be generated by decreased MMRin bacterial strains, we induced the TACLACPMS134-pTLACZ cells for 48hours with IPTG as described above. We then diluted the culture 1:50 inLB medium, grew the strain for 24 hours, and plated 10 μls of culture(diluted in 300 μls of LB) onto LB amp-XGAL plates to identify candidateclones that produce robust levels of recombinant LACZ protein. As acontrol, uninduced cells were treated similarly and plated onto LBamp-XGAL plates. Analysis of the plates revealed a number of bacterialcolonies exhibiting a number of clones with an intense BLUE staining inthe TACLACPMS134/pTLACZ cells induced with IPTG but none were observedin uninduced clones (FIG. 6). To confirm that these clones produced anenhanced level of LACZ, we expanded 2 clones with an average BLUE stain(BGAL-C1 and BGAL-C2) and 10 clones with a robust BLUE staining(BGAL-MOR1 to BGAL-MOR10). We grew all clones in LB AMP for 24 hourswithout IPTG and replated the clones. Six out of ten BGAL-MOR clonesresulted in a more robust β-gal stain in situ as compared to control“average” cells (BGALC1 and C2). We next performed a more quantitativeassay using a β-gal ELISA assay. Briefly, 2 mls of cell centrifuged at10,000 gs for 10 minutes and resuspended in 0.5 mls of 0.25M Tris, pH7.5 plus 0.0001% Tween-20. Cells were freeze-thawed 4×'s and vortexedfor 4 minutes at room temperature. Lysates were cleared of debris bycentrifugation and supernatants were collected. Protein extracts werequantified for total protein using the Bradford assay (BioRad) asdescribed by the manufacturer. Plate ELISAs were carried out by coating96 well maxisorb (NUNC) plates with 0.1 mls of a 1 μg/ml (diluted in PBSpH7.0) bacterial extract solution and a dose range of recombinant β-GAL(Sigma) from 0.001 to 10 mg/ml. All samples were plated in triplicates.Plates were coated for 2 hours, washed 2 times with PBS and blocked with0.2 mls of PBS plus 5% powdered milk for 30 minutes. Next, plates werewashed once with PBS and incubated with an anti-β3-galactosidasemonoclonal antibody that recognizes both native and denatured forms(Sigma) for 2 hours. Plates were then washed 3 times with PBS andincubated with 0.1 mls of an anti-mouse horseradish peroxidaseconjugated antibody for 1 hour at room temperature. Plates were washed 3times with PBS and incubated with TMB ELISA substrate (BioRad) for 15 to30 minutes. Reactions were stopped with 0. IN H₂SO₄ and read on a BioRADplate reader at 415 nm. The control clones produced roughly 9 and 13 μgs/ml of β-gal while BGAL-MOR clones 2, 3 and 9 produced 106, 82 and 143μgs /ml of β-gal. To determine if reason that these clones produced moreβ-gal was due to mutations in the plasmid promoter elements, we isolatedthe pTLACZ plasmid and retransfected it into DH10B cells as describedabove. In situ analysis found the resultant clones to produce similaramount of β-gal as that of the control. These data suggest that theBGAL-MOR 2, 3, and 9 hosts had alterations, which results in elevatedexpression and/or stability of recombinant proteins.

To determine if the enhanced in situ β-gal expression that was observedin BGAL-MOR clones 1, 5, and 6, which did not appear to have enhancedβ-gal protein levels (had less than 15 μg/ml as determined by ELISA) wasauthentic, we performed a more quantitative assay on these lines plusthe BGAL-MOR 9, the BGALC1 and C2 lines as control. Cells containing anempty vector (without a LACZ gene) were used as negative control. Tomeasure β-gal activity, we employed a calorimetric β-gal substrate assayusing CPRG (Roche) as described (31). Briefly, 5 μgs of protein extractisolated for ELISA analysis (described above) were analyzed using aplate assay. Protein was added to buffer containing 45 mM2-mercaptoethanol, 1 mM MgCl₂, 0.1 M NaPO₄ and 0.6 mg/ml Chlorophenolred-β-D-galactopyranoside (CPRG, Roche). Reactions were incubated for 1hour, terminated by the addition of 0.5 M Na₂CO₃, and analyzed byspectrophotometry at 576 nm in a BioRad plate reader. Analysis of theseextracts confirmed our in situ data that these cells did have increasedβ-gal activity (TABLE 3).

TABLE 3 Generation of bacterial clones with increased β-gal enzymaticactivity via MMR deficiency. β-gal protein β-gal activity Clone (μg/ml)(O.D. 576) BGAL-C1  9 0.413 +/− .092 BGAL-C2  13 0.393 +/− .105BGAL-MOR1  14 0.899 +/− .134 BGAL-MOR5  13 0.952 +/− .133 BGAL-MOR6  150.923 +/− .100 BGAL-MOR9 143 0.987 +/− .106 Empty vector — 0.132 +/−.036

Because there was no observable increase in the amount of β-gal proteinone likely hypothesis is that the β-gal gene structure was mutatedduring the hypermutability growth stage and now produces a more activeenzyme. Sequence analysis confirms that this may be the reason forenhanced activity in a subset of clones.

Together, these data demonstrate the ability to produce geneticallyaltered prokaryotic host strains using dominant negative MMR genes togenerate commercially valuable output traits such as high recombinantprotein producer lines and structurally altered enzymes with enhancedactivities.

EXAMPLE 4 Mutations in the Host Genome Generated by Defective MMR areGenetically Stable

As described in EXAMPLE 2 and 3, manipulation of the MMR pathway inmicrobes results in alterations within the host genome and the abilityto select for a novel output traits. It is important that the mutationsintroduced as a result of defective MMR is genetically stable and passedon to daughter cells once a desired output pathway is established. Todetermine the genetic stability of mutations introduced into themicrobial genome the following experiment was performed. Fiveindependent colonies from pT7-PMS 134 and pT7-PMSR3 that are kanamycinresistance were grown overnight from an isolated colony in 5 ml of LB.Next, 1 μL of the overnight culture from these cultures were inoculatedinto another 5 mL of LB and grown overnight to saturation. Under thesegrowth conditions the microbial cells have divided over 20 generations.Therefore, if the new output trait generated by alteration of MMR isunstable, the cells should “revert” back from kanamycin resistance tokanamycin sensitivity. Cells were plated onto LB plates and incubatedovernight at 37° C. Next, the colonies (about 1,000/plate) were replicaplated to LB, LB^(amp100), and LB^(kan50) plates and incubated at 37° C.overnight. Analysis of clones from these studies reveal that a strictcorrelation occurs with loss of dominant negative MMR expression andphenotype stability. No loss of KAN resistant clones generated inExample 3 were observed when cells were grown in the absence of IPTG(not expressing PMS134), while 5 revertants out of 1200 were observed inclones the were continually grown in IPTG (express PMS134). Extendedculturing of cells and replica plating found no reversions of KANresistance in cultures grown in the absence of IPTG, which produce noPMS134 as determined by western blot (data not shown).

These data demonstrate the utility of employing inducible expressionsystems and dominant negative MMR genes in prokaryotes to generategenetically altered strains for commercial applications such as but notlimited to enhanced recombinant manufacturing and biotransformation thatcan then in turn be restored to a genetically stable host with a “fixed”new genotype that is suitable for commercial processes.

EXAMPLE 5 Enhanced Generation of MMR-Defective Bacteria and ChemicalMutagens for the Generation of New Output Traits

It has been previously documented that MMR deficiency yields toincreased mutation frequency and increased resistance to toxic effectsof chemical mutagens (CM) and their respective analogues such as but notlimited to those as: ethidium bromide, EMS, MNNG, MNU, Tamoxifen,8-Hydroxyguanine, as well as others listed but not limited to inpublications by: Khromov-Borisov, N. N., et. al. (Mutat. Res. 430:55–74,1999); Ohe, T., et. al. (Mutat. Res. 429:189–199, 1999); Hour, T. C. et.al. (Food Chem. Toxicol. 37:569–579, 1999); Hrelia, P., et. al. (Chem.Biol. Interact. 118:99–111, 1999); Garganta, F., et. al. (Environ. Mol.Mutagen. 33:75–85, 1999); Ukawa-Ishikawa S., et. al. (Mutat. Res.412:99–107, 1998); the website having the URL address: www. host server,ehs.utah.edu domain name, ohh directory, mutagens subdirectory, etc. Todemonstrate the ability of CMs to increase the mutation frequency in MMRdefective bacterial cells, we exposed T7-PMS134 BL21 cells to CMs.

T7-PMS134 cells and empty vector control cells were grown with IPTG for48 hours and then diluted 1:50 in LB plus IPTG and increasing amounts ofethyl methane sulfonate (EMS) from 0, 1, 10, 50, 100, and 200 μM. 10 μLaliquots of culture (diluted in 300 μl LB) were plated out on LB agarplus ampicillin plates and grown overnight at 37C. The next day plateswere analyzed for cell viability as determined by colony formation.Analysis found that while no significant difference in colony number wasobserved between the pT7-PMS134 and control at the 0, 1 , or 10 μMconcentrations (all had >1000 colonies), the number of control cellswere reduced to 30 and 0 at the 50 and 100 μM concentrations,respectively. No difference was observed in the pT7-PMS134 cells treatedwith 0, 1, 10 or 50 μM, while a 3 fold reduction was observed incultures treated with 100 μM EMS. The 200 μM treatment was toxic forboth lines. These data demonstrate the ability of MMR deficiency toprotect prokaryotes against the toxic effects of DNA akylating agentsand provides a means to generate a wider range of mutations that canlead to an increased number of genetic variations and an increase in thenumber of new biochemical activities within host proteins to produce newoutput traits for commercial applications.

To confirm that MMR deficient bacterial cells treated with CM can resultin an increased mutation rate and produce a greater number of variants,we cultured pT7-PMS134 cells and empty vector controls in the presenceof IPTG for 48 hours, followed by dilution and regrowth in 25 μM EMS for24 hours as described above. Cells were plated out on 100 mM petridishes containing amplicillin or KAN and scored for KAN resistance.Analysis revealed that an 11-fold increase in the generation of KANresistant cells were found in pT7-Ea-PMS134V5 cells in contrast tocontrol cells.

These data demonstrate the use of employing a regulated dominantnegative MMR system plus chemical mutagens to produce enhanced numbersof genetically altered prokaryotic strains that can be selected for newoutput traits. This methods is now useful generating such organisms forcommercial applications such as but not limited to recombinantmanufacturing, biotransformation, and altered biochemicals(biotransformation) with enhanced activities for manufacturing purposesand gene discovery for pharmaceutical compound development.

EXAMPLE 6 Alternative Methods to Inhibition of Bacterial MMR Activity

The inhibition of MMR activity in a host organism can be achieved byintroducing a dominant negative allele as shown in EXAMPLES 2 and 3.This application also teaches us the use of using regulated systems tocontrol MMR in prokaryotes to generate genetic diversity and outputtraits for commercial applications. Other ways to regulate thesuppression of MMR activity of a host is by using genetic recombinationto knock out alleles of a MMR gene that can be spliced out such afterselection using a system such as the CRE-Lox system; 2) blocking MMRprotein dimerization with other subunits (which is required foractivity) by the introduction of polypeptides or antibodies into thehost via transfection methods routinely used by those skilled in theart; or 3) decreasing the expression of a MMR gene using anti-senseoligonucleotides.

MMR gene knockouts. We intend to generate disrupted targeting vectors ofa particular MMR gene and introduce it into the genome of bacteria usingmethods standard in the art. Bacteria exhibiting hypermutability will beuseful to produce genetically diverse offspring for commercialapplications. Bacteria will be confirmed to have lost the expression ofthe MMR gene using standard northern and biochemical techniques (asdescribed in reference 32). MMR gene loci can be knocked out, strainsselected for new output traits and MMR restored by introducing awildtype MMR gene to complement the KO locus. Other strategies includeusing KO vectors that can target a MMR gene locus, select for hostoutput traits and then have the KO vector “spliced” from the genomeafter strain generation. This process could be performed using systemssuch as but not limited to CRE-Lox.Blocking peptides. MMR subunits (MutS and MutL proteins) interact toform active MMR complexes. Peptides are able to specifically inhibit thebinding of two proteins by competitive inhibition. The use of peptidesor antibodies to conserved domains of a particular MMR gene can beintroduced into prokaryotic cells using lipid transfer methods that arestandard in the art. Bacteria will be confirmed to have lost theexpression of the MMR gene using standard northern and biochemicaltechniques (as described in reference 32). Bacteria exhibitinghypermutability will be useful to produce genetically diverse sibs forcommercial applications.Discussion

The results described above will lead to several conclusions. Theexpression of dominant negative MMR proteins results in an increase inhypermutability in bacteria. This activity is due to the inhibition ofMMR biochemical activity in these hosts. This method provides a claimfor use of dominant negative MMR genes and their encoded products forthe creation of hypermutable bacteria to produce new output traits forcommercial applications.

Examples of MMR Genes and Encoded Polypeptides

Yeast MLH1 cDNA (accession number U07187) (SEQ ID NO: 5) 1 aaataggaatgtgatacctt ctattgcatg caaagatagt gtaggaggcg ctgctattgc 61 caaagacttttgagaccgct tgctgtttca ttatagttga ggagttctcg aagacgagaa 121 attagcagttttcggtgttt agtaatcgcg ctagcatgct aggacaattt aactgcaaaa 181 ttttgatacgatagtgatag taaatggaag gtaaaaataa catagaccta tcaataagca 241 atgtctctcagaataaaagc acttgatgca tcagtggtta acaaaattgc tgcaggtgag 301 atcataatatcccccgtaaa tgctctcaaa gaaatgatgg agaattccat cgatgcgaat 361 gctacaatgattgatattct agtcaaggaa ggaggaatta aggtacttca aataacagat 421 aacggatctggaattaataa agcagacctg ccaatcttat gtgagcgatt cacgacgtcc 481 aaattacaaaaattcgaaga tttgagtcag attcaaacgt atggattccg aggagaagct 541 ttagccagtatctcacatgt ggcaagagtc acagtaacga caaaagttaa agatgacaga 601 tgtgcatggagagtttcata tgcagaaggt aagatgttgg aaagccccaa acctgttgct 661 ggaaaagacggtaccacgat cctagttgaa gacctttttt tcaatattcc ttctagatta 721 agggccttgaggtcccataa tgatgaatac tctaaaatat tagatgttgt cgggcgatac 781 gccattcattccaaggacat tggcttttct tgtaaaaagt tcggagactc taattattct 841 ttatcagttaaaccttcata tacagtccag gataggatta ggactgtgtt caataaatct 901 gtggcttcgaatttaattac ttttcatatc agcaaagtag aagatttaaa cctggaaagc 961 gttgatggaaaggtgtgtaa tttgaatttc atatccaaaa agtccatttc attaattttt 1021 ttcattaataatagactagt gacatgtgat cttctaagaa gagctttgaa cagcgtttac 1081 tccaattatctgccaaaggg cttcagacct tttatttatt tgggaattgt tatagatccg 1141 gcggctgttgatgttaacgt tcacccgaca aagagagagg ttcgtttcct gagccaagat 1201 gagatcacagagaaaatcgc caatcaattg cacgccgaat tatctgccat tgatacttca 1261 cgtactttcaaggcttcttc aatttcaaca aacaagccag agtcattgat accatttaat 1321 gacaccatagaaagtgatag gaataggaag agtctccgac aagcccaagt ggtagagaat 1381 tcatatacgacagccaatag tcaactaagg aaagcgaaaa gacaagagaa taaactagtc 1441 agaatagatgcttcacaagc taaaattacg tcatttttat cctcaagtca acagttcaac 1501 tttgaaggatcgtctacaaa gcgacaactg agtgaaccca aggtaacaaa tgtaagccac 1561 tcccaagaggcagaaaagct gacactaaat gaaagcgaac aaccgcgtga tgccaataca 1621 atcaatgataatgatttgaa ggaccaacct aagaagaaac aaaagttggg ggattataaa 1681 gttccaagcattgccgatga cgaaaagaat gcactcccga tttcaaaaga cgggtatatt 1741 agagtacctaaggagcgagt taatgttaat cttacgagta tcaagaaatt gcgtgaaaaa 1801 gtagatgattcgatacatcg agaactaaca gacatttttg caaatttgaa ttacgttggg 1861 gttgtagatgaggaaagaag attagccgct attcagcatg acttaaagct ttttttaata 1921 gattacggatctgtgtgcta tgagctattc tatcagattg gtttgacaga cttcgcaaac 1981 tttggtaagataaacctaca gagtacaaat gtgtcagatg atatagtttt gtataatctc 2041 ctatcagaatttgacgagtt aaatgacgat gcttccaaag aaaaaataat tagtaaaata 2101 tgggacatgagcagtatgct aaatgagtac tattccatag aattggtgaa cgatggtcta 2161 gataatgacttaaagtctgt gaagctaaaa tctctaccac tacttttaaa aggctacatt 2221 ccatctctggtcaagttacc attttttata tatcgcctgg gtaaagaagt tgattgggag 2281 gatgaacaagagtgtctaga tggtatttta agagagattg cattactcta tatacctgat 2341 atggttccgaaagtcgatac actcgatgca tcgttgtcag aagacgaaaa agcccagttt 2401 ataaatagaaaggaacacat atcctcatta ctagaacacg ttctcttccc ttgtatcaaa 2461 cgaaggttcctggcccctag acacattctc aaggatgtcg tggaaatagc caaccttcca 2521 gatctatacaaagtttttga gaggtgttaa ctttaaaacg ttttggctgt aataccaaag 2581 tttttgtttatttcctgagt gtgattgtgt ttcatttgaa agtgtatgcc ctttccttta 2641 acgattcatccgcgagattt caaaggatat gaaatatggt tgcagttagg aaagtatgtc 2701 agaaatgtatattcggattg aaactcttct aatagttctg aagtcacttg gttccgtatt 2761 gttttcgtcctcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gtaccaaaga 2821 cccgagtccttttatgagag aaaacatttc atcatttttc aactcaatta tcttaatatc 2881 attttgtagtattttgaaaa caggatggta aaacgaatca cctgaatcta gaagctgtac 2941 cttgtcccataaaagtttta atttactgag cctttcggtc aagtaaacta gtttatctag 3001 ttttgaaccgaatattgtgg gcagatttgc agtaagttca gttagatcta ctaaaagttg 3061 tttgacagcagccgattcca caaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt 3121 aatggtttgcatcaccatcg gatgtctgtt gaaaaactca ctttttgcat ggaagttatt 3181 aacaataagactaacgatta ccttagaata atgtataa Yeast MLH1 protein (accession numberU07187) (SEQ ID NO: 15) MSLRIKALDASVVNKIAAGEIIISPVNALKEMMENSIDANATMIDILVKEGGIKVLQITDNGSGINKADLPILCERFTTSKLQKFEDLSQIQTYGFRGEALASISHVARVTVTTKVKEDRCAWRVSYAEGKMLESPKPVAGKDGTTILVEDLFFNIPSRLRALRSHNDEYSKILDVVGRYAIHSKDIGFSCKKFGDSNYSLSVKPSYTVQDRIRTVFNKSVASNLITFHISKVEDLNLESVDGKVCNLNFISKKSISLIFFINNRLVTCDLLRRALNSVYSNYLPKGFRPFIYLGIVIDPAAVDVNVHPTKREVRFLSQDEIIEKIANQLHAELSAIDTSRTFKASSISTNKPESLIPFNDTIESDRNRKSLRQAQVVENSYTTANSQLRKAKRQENKLVRIDASQAKITSFLSSSQQFNFEGSSTKRQLSEPKVTNVSHSQEAEKLTLNESEQPRDANTINDNDLKDQPKKKQKLGDYKVPSIADDEKNALPISKDGYIRVPKERVNVNLTSIKKLREKVDDSIHRELTDIFANLNYVGVVDEERRLAAIQHDLKLFLIDYGSVCYELFYQIGLTDFANFGKINLQSTNVSDDIVLYNLLSEFDELNDDASKEKIISKIWDMSSMLNEYYSIELVNDGLDNDLKSVKLKSLPLLLKGYIPSLVKLPFFIYRLGKEVDWEDEQECLDGILREIALLYIPDMVPKVDTLDASLSEDEKAQFINRKEHISSLLEHVLFPCIKRRFLAPRHILKDVVEIANLPDLYKVFERC Mouse PMS2 protein (SEQ ID NO: 16)MEQTEGVSTE CAKAIKPIDG KSVHQICSGQ VILSLSTAVK ELIENSVDAG ATTIDLRLKD 60YGVDLIEVSD NGCGVEEENF EGLALKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120TISTCHGSAS VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE 180YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SCMKENIGSV FGQKQLQSLI 240PFVQLPPSDA VCEEYGLSTS GRHKTFSTFR ASFHSARTAP GGVQQTGSFS SSIRGPVTQQ 300RSLSLSMRFY HMYNRHQYPF VVLNVSVDSE CVDINVTPDK RQILLQEEKL LLAVLKTSLI 360GMFDSDANKL NVNQQPLLDV EGNLVKLHTA ELEKPVPGKQ DNSPSLKSTA DEKRVASISR 420LREAFSLHPT KEIKSRGPET AELTRSFPSE KRGVLSSYPS DVISYRGLKG SQDKLVSPTD 480SPGDCMDREK IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL 540DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA 600EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC PGENQAAEDE 660LRKEISKSMF AEMEILGQFN LGFIVTKLKE DLFLVDQHAA DEKYNFEMLQ QHTVLQAQRL 720ITPQTLNLTA VNEAVLIENL EIFRKNGFDF VIDEDAPVTE RAKLISLPTS KNWTFGPQDI 780DELIFMLSDS PGVMCRPSRV RQMFASRACR KSVMIGTALN ASEMKKLITM MGEMDHPWNC 840PHGRPTMRHV ANLDVISQN 859 Mouse PMS2 cDNA (SEQ ID NO: 6) gaattccggtgaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcgtcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120 gtcttttcccgagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg 180 catccatggagcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240 atgggaagtcagtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg 300 tgaaggagttgatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta 360 aagactatggggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaaggtctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480 cgcaggttgaaactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg 540 atgtcactatatctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600 ataatgggaaaatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg 660 tgcagcacttattttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa 720 aggagtattccaaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctgcactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctggcatgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttttgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacgccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggaggagtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtctccaagcttg tctatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgtccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggcaaattctacta caagaagaga agctattgct ggccgtttta aagacctcct 1260 tgataggaatgtttgacagt gacgcaaaca agcttaatgt caaccagcag ccactgctag 1320 atgttgaaggtaacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa 1380 agcaagataactctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct 1440 ccaggctgagagaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500 agactgctgaactgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgtcatctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccctggtgactgt atggacagag agaaaataga aaaagactta gggctcagca 1680 gcacctcagctggctctgag gaagagttca gcaccccaga agtggccagt agctttagca 1740 gtgactataacgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg 1800 acctggactgccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagctctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagaccctcaaatgtc aacattcctc aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggtcgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040 tgagttctctagctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg 2100 aactgagttacagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcagaaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctgggatttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280 ctgcggatgagaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcacaccccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaatattcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca 2460 ctgaaagggctaaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatgaactgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagacagatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgagcgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga 2700 actgcccccacggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga 2760 actgacacaccccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaatctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc 2880 catttaaaagcagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtgggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000 agactcaattcaaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056 human PMS2protein (SEQ ID NO: 17) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSVDVKLENYGFD KIEVRDNGEG 60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSICCIAEVLITT RTAADNFSTQ 120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYSTAKKCKDEIK KIQDLLMSFG 180 ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMNNMESFQYHSE ESQIYLSGFL 240 PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHYNLKCLKESTR LYPVFFLKID 300 VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPLPSTNSYENNK TDVSAADIVL 360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESGKNTDDCLNHQ ISIGDFGYGH 420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFISSVKHTQSENG NKDHIDESGE 480 NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVPEKSLPCKVSN NNYPIPEQMN 540 LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASALFVQDHRPQFL IENPKTSLED 600 ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIEQESQMSLKDG RKKIKPTSAW 660 NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIPFSMKNLKINF KKQNKVDLEE 720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKRLLENHKLPAE PLEKPIMLTE 780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKIKLIPGVSITE NYLEIEGMAN 840 CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGEAVRLSRQLPM YLSKEDIQDI 900 IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT 932 HumanPMS2 cDNA (SEQ ID NO: 7) cgaggcggat cgggtgttgc atccatggag cgagctgagagctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcagatttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaacagtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatcttattgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctgaaacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggctttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgccacgcatcggc gaaggttgga 420 actcgactga tgtttgatca caatgggaaa attatccagaaaacccccta cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta tttcccacactacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtccaggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc accaatcagcttggacaagg aaaacgacag 660 cctgtggtac gcacaggtgg aagccccagc ataaaggaaaatatcggctc tgtgtttggg 720 cagaagcagt tgcaaagcct cattcctttt gttcagctgccccctagtga ctccgtgtgt 780 gaagagtacg gtttgagctg ttcggatgct ctgcataatcttttttacat ctcaggtttc 840 atttcacaat gcacgcatgg agttggaagg agttcaacagacagacagtt tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcgtgaatgaggt ctaccacatg 960 tataatcgac accagtatcc atttgttgtt cttaacatttctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga taaaaggcaa attttgctacaagaggaaaa gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtgatgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt aacttaataaaaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcattaaggactgg agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctcttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca agaaggagccctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaaggcgtcctgag acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacggacagagcgga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat tctgaggggttcagcatccc agacacgggc 1560 agtcactgca gcagcgagta tgcggccagc tccccaggggacaggggctc gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctttttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt cgagttttgcctcagccaac taatctcgca 1740 accccaaaca caaagcgttt taaaaaagaa gaaattctttccagttctga catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttgatgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg agttctttagctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag tgaaggggaa cagaattacaggaagtttag ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaaaagagataag taaaacgatg 2040 tttgcagaaa tggaaatcat tggtcagttt aacctgggatttataataac caaactgaat 2100 gaggatatct tcatagtgga ccagcatgcc acggacgagaagtataactt cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcacctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat ctggaaatatttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc tccagtcact gaaagggctaaactgatttc cttgccaact 2340 agtaaaaact ggaccttcgg accccaggac gtcgatgaactgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga gtcaagcagatgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcgagatgaagaa actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatggaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac tgaccgtagtcactgtatgg aataattggt 2640 tttatcgcag atttttatgt tttgaaagac agagtcttcactaacctttt ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccatttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 human PMS1 protein (SEQ IDNO: 18) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFDKIEVRDNGEG 60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITTRTAADNFSTQ 120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS TAKKCKDEIKKIQDLLMSFG 180 ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMN NMESFQYHSEESQIYLSGFL 240 PKCDADHSFT SLSTPERSFI FTNSRPVHQK DILKLIRHHY NLKCLKESTRLYPVFFLKID 300 VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNKTDVSAADIVL 360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQISIGDFGYGH 420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS SVKHTQSENGNKDHIDESGE 480 NEESAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP EKSLPCKVSNNNYPIPEQMN 540 LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL FVQDHRPQFLIENPKTSLED 600 ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDGRKKIKPTSAW 660 NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSMKNLKINFKKQNKVDLEE 720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAEPLEKPIMLTE 780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI KLIPGVSITENYLEIEGNAN 840 CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE AVRLSRQLPNYLSKEDIQDT 900 IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT 932 Human PMS1 cDNA(SEQ ID NO: 8) ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgctagcagcaag 60 ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacaptccgactcctttcaa 120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaaaactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgataaaattgagg 240 tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggcaatgaagtact 300 acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttacggttttcgtg 360 gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaacaagaacggctg 420 ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactttctcagaaac 480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaagaatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaaaagatccaag 600 atctcctcat gagctttggt atccttaaac ctgacttaag gattgtctttgtacataaca 660 aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctcatgtcagttc 720 tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaagaatctcaga 780 tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcactagtctttcaa 840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaagatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgtttgtatcctg 960 ttttttttct gaaaatcgat gttcctacag ctgatgttga tgtaaatttaacaccagata 1020 aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaaaatctgatga 1080 cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaaacagatgttt 1140 ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgctttttaataaagtgg 1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattccaaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccagataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaaaacactaaga 1380 atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcagacggaatata 1440 gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggcaataaagacc 1500 atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactcttcggaaattt 1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagagaatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaataataattatc 1680 caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatcaaatgtaatag 1740 ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagtaatcaagaaac 1800 ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctcatagaaaatc 1860 ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaagacattgagtg 1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacgatacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggcagaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacctcattatcta 2100 atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaaggagtcaaaata 2160 ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaattttaagaaacaaa 2220 acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctcaggtttcctg 2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatatagagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagagccactggaaa 2400 agccaattat gttaacagag agtcttttta atggatctca ttatttagacgttttatata 2460 aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgatcctcgtctta 2520 cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaaaattacttgg 2580 aaatagaagg aatggctaat tgtctcccat tctatggagt agcagatttaaaagaaattc 2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacctcgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatgtacttatcaa 2760 aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaatgaaattaaag 2820 agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaaactacatgat 2880 taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaaacagcatgag 2940 tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaatgaggattca 3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaaataaactaat 3060 aac 3063 human MSHz protein (SEQ ID NO: 19) NAVQPKETLQLESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHDEDA LLAAREVFKT 60 QGVIKYMGPAGAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA 120 YKASPGNLSQFEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ RKLGLCEFPD 180 NDQFSNLEALLIQIGPKECV LPGGETAGDM GKLRQIIQRG GILITERKKA DFSTKDIYQD 240 LNRLLKGKKGEQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM 300 KLDIAAVRALNLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL MDKNRIEERL 360 NLVEAFVEDAELRQTLQEDL LRRFPDLNRL AKKFQRQAAN LQDCYRLYQG INQLPNVIQA 420 LEKHEGKHQKLLLAVFVTPL TDLRSDFSKF QEMIETTLDM DQVENHEFLV KPSFDPNLSE 480 LREIMNDLEKKMQSTLISAA RDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS 540 TVDIQKNGVKFTNSKLTSLN EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA 600 QLDAVVSFAHVSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD 660 KQMFHIITGPNMGGKSTYIR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK 720 GVSTFMAEMLETASILRSAT KDSLIIIDEL GRGTSTYDGF GLAWAISEYI ATKIGAFCMF 780 ATHFHELTALANQIPTVNNL HVTALTTEET LTMLYQVKKG VCDQSEGIHV AELANFPKHV 840 IECAKQKALELEEFQYIGES QGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS 900 EENITIKLKQLKAEVIAKNN SFVNEIISRI KVTT 934 Human MSH2 cDNA (SEQ ID NO: 9) ggcgggaaacagcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacatggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120 gcttcgtgcgcttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg 180 accggggcgacttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt 240 tcaagacccagggggtgatc aagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttagtaaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttgaagtttataag aatagagctg gaaataaggc atccaaggag aatgattggt 420 atttggcatataaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta 480 acaatgatatgtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc 540 agagacaggttggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat 600 tccctgataatgatcagttc tccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgttttacccggagga gagactgctg gagacatggg gaaactgaga cagataattc 720 aaagaggaggaattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt 780 atcaggacctcaaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840 tgccagaaatggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag 900 aactcttatcagatgattcc aactttggac agtttgaact gactactttt gacttcagcc 960 agtatatgaaattggatatt gcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccactggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagacttgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaatttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttacttcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag 1260 cagcaaacttacaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta 1320 tacaggctctggaaaaacat gaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttactgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatggatcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaattaagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccagagatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac 1620 agtttggatattactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtactgtagatatc cagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatgaagagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaattgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctcagctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacgaccagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttgtgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataaacagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgacaaactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcagaagtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaaggagtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaaagattcatta ataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgggttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgcaacccatttt catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctacatgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgtctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaatagagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgcaaggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtgaaaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcagaagaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatagctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatggaatgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccctttttccata gtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagtaatattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactgaggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaatcatgtagtt tgtgg 3145 human MLH1 protein (SEQ ID NO: 20) MSFVAGVIRRLDETVVNRIA AGEVIQRPAN AIKEMIENCL DAKSTSIQVI VKEGGLKLIQ 60 IQDNGTGIRKEDLDIVCERF TTSKLQSFED LASISTYGFR GEALASISHV AHVTITTKTA 120 DGKCAYRASYSDGKLKAPPK PCAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV 180 GRYSVHNAGISFSVKKQGET VADVRTLDNA STVDNIRSIF GNAVSRELIE IGCEDKTLAF 240 KMNGYISNANYSVKKCIFLL FINHRLVEST SLRKAIETVY AAYLPKNTHP FLYLSLEISP 300 QNVDVNVHPTKHEVHFLHEE SILERVQQHI ESKLLGSNSS RMYFTQTLLP GLAGPSGEMV 360 KSTTSLTSSSTSGSSDKVYA NQMVRTDSRE QKLDAFLQPL SKPLSSQPQA IVTEDKTDIS 420 SGRARQQDEEMLELPAPAEV AAKNQSLEGD TTKGTSEMSE KRGPTSSNPR KRHREDSDVE 480 MVEDDSRKEMTAACTPRRRI INLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL 540 AQHQTKLYLLNTTKLSEELF YQILIYDFAN FGVLRLSEPA PLFDLAMLAL DSPESGWTEE 600 DGPKEGLAEYIVEFLKKKAE MLADYFSLEI DEEGNLIGLP LLIDNYVPPL EGLPIFTLRL 660 ATEVNWDEEKECFESLSKEC AMFYSIRKQY ISEESTLSGQ QSEVPGSIPN SWKWTVEHIV 720 YKALRSHILPPKHFTEDGNI LQLANLPDLY KVFERC 756 Human MLH1 cDNA (SEQ ID NO: 10)cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag 60acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa 120gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt 300atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt 360actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga 420aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag 480gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgtaggcat tagtttctca 600gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg 660gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt 720gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg 780aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga 840aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt 1200gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag 2100gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata 2460aataaataga tgtgtcttaa cata 2484 hPMS2-134 protein (SEQ ID NO: 21)MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG 60IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ 120YVLDGSGHIL SQK 133 hPMS2-134 cDNA (SEQ ID NO: 11) cgaggcggat cgggtgttgcatccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattgatcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggtaaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaaggactatgga gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaacttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaactcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcgatgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 hMSH6 (humancDNA) ACCESSION U28946 (SEQ ID NO: 22)MSRQSTLYSFFPKSPALSDANKASARASREGGRAAAAPGASPSPGGDAAWSEAGPGPRPLARSASPPKAKNLNGGLRRSVAPAAPTSCDFSPGDLVWAKMEGYPWWPCLVYNHPFDGTFIREKGKSVRVHVQFFDDSPTRGWVSKRLLKPYTGSKSKEAQKGGHFYSAKPEILRAMQRADEALNKDKIKRLELAVCDEPSEPEEEEEMEVGTTYVTDKSEEDNEIESEEEVQPKTQGSRRSSRQIKKRRVISDSESDIGGSDVEFKPDTKEEGSSDEISSGVGDSESEGLNSPVKVARKRKRMVTGNGSLKRKSSRKETPSATKQATSISSETKNTLRAFSAPQNSESQAHVSGGGDDSSRPTVWYHETLEWLKEEKRRDEHRRRPDHPDFDASTLYVPEDFLNSCTPGMRKWWQIKSQNFDLVICYKVGKFYELYHNDALIGVSELGLVFMKGNWAHSGFPEIAFGRYSDSLVQKGYKVARVEQTETPEMMEARCRKMAHISKYDRVVRREICRIITKGTQTYSVLEGDPSENYSKYLLSLEKKEEDSSGHTRAYGVCFVDTSLGKFFIGQFSDDRNCSRFRTLVAHYPPVQVLFEKGNLSKETKTILKSSLSCSLQEGLIPGSQFWDASKTLRTLLEEEYFREKLSDGIGVMLPQVLKGMTSESDSIGLTPGEKSELALSALGGCVFYLKKCLIDQELLSMANFEEYIPLDSDTVSTTRSGAIFTKAYQRMVLDAVTLNNLEIFLNGTNGSTEGTLLERVDTCHTPFGKRLLKQWLCAPLCNHYAINDRLDAIEDLMVVPDKISEVVELLKKLPDLERLLSKIHNVGSPLKSQNHPDSRAIMYEETTYSKKKIIDFLSALEGFKVMCKIIGIMEEVADGFKSKILKQVISLQTKNPEGRFPDLTVELNRWDTAFDHEKARKTGLITPKAGFDSDYDQALADIRENEQSLLEYLEKQRNRIGCRTIVYWGIGRNRYQLEIPENFTTRNLPEEYELKSTKKGCKRYWTKTIEKKLANLTNAEERRDVSLKDCMRRLEYNFDKNYKDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVILLPEDTPPFLELKGSRHPCITKTFFGDDFIPNDILIGCEEEEQENGKAYCVLVTGPNMGGKSTLMRQAGLLAVMAQMGCYVPAEVCRLTPIDRVFTRLGASDRIMSGESTFFVELSETASILMHATAHSLVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNVAVRLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGHRKAREFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL” hPMSR2 (human cDNA) ACCESSIONU38964 (SEQ ID NO: 12) 1 ggcgctccta cctgcaagtg gctagtgcca agtgctgggccgccgctcct gccgtgcatg 61 ttggggagcc agtacatgca ggtgggctcc acacggagaggggcgcagac ccggtgacag 121 ggctttacct ggtacatcgg catggcgcaa ccaaagcaagagagggtggc gcgtgccaga 181 caccaacggt cggaaaccgc cagacaccaa cggtcggaaaccgccaagac accaacgctc 241 ggaaaccgcc agacaccaac gctcggaaac cgccagacaccaaggctcgg aatccacgcc 301 aggccacgac ggagggcgac tacctccctt ctgaccctgctgctggcgtt cggaaaaaac 361 gcagtccggt gtgctctgat tggtccaggc tctttgacgtcacggactcg acctttgaca 421 gagccactag gcgaaaagga gagacgggaa gtattttttccgccccgccc ggaaagggtg 481 gagcacaacg tcgaaagcag ccgttgggag cccaggaggcggggcgcctg tgggagccgt 541 ggagggaact ttcccagtcc ccgaggcgga tccggtgttgcatccttgga gcgagctgag 601 aactcgagta cagaacctgc taaggccatc aaacctattgatcggaagtc agtccatcag 661 atttgctctg ggccggtggt accgagtcta aggccgaatgcggtgaagga gttagtagaa 721 aacagtctgg atgctggtgc cactaatgtt gatctaaagcttaaggacta tggagtggat 781 ctcattgaag tttcaggcaa tggatgtggg gtagaagaagaaaacttcga aggctttact 841 ctgaaacatc acacatgtaa gattcaagag tttgccgacctaactcaggt ggaaactttt 901 ggctttcggg gggaagctct gagctcactt tgtgcactgagtgatgtcac catttctacc 961 tgccgtgtat cagcgaaggt tgggactcga ctggtgtttgatcactatgg gaaaatcatc 1021 cagaaaaccc cctacccccg ccccagaggg atgacagtcagcgtgaagca gttattttct 1081 acgctacctg tgcaccataa agaatttcaa aggaatattaagaagaaacg tgcctgcttc 1141 cccttcgcct tctgccgtga ttgtcagttt cctgaggcctccccagccat gcttcctgta 1201 cagcctgtag aactgactcc tagaagtacc ccaccccacccctgctcctt ggaggacaac 1261 gtgatcactg tattcagctc tgtcaagaat ggtccaggttcttctagatg atctgcacaa 1321 atggttcctc tcctccttcc tgatgtctgc cattagcattggaataaagt tcctgctgaa 1381 aatccaaaaa aaaaaaaaaa aaaaaaaa hPMSR2 (humanprotein) ACCESSION U38964 (SEQ ID NO: 23)MAQPKQERVARARHQRSETARHQRSETAKTPTLGNRQTPTLGNRQTPRLGIHARPRRRATTSLLTLLLAFGKNAVRCALIGPGSLTSRTRPLTEPLGEKERREVFFPPRPERVEHNVESSRWEPRRRGACGSRGGNFPSPRGGSGVASLERAENSSTEPAKAIKPIDRKSVHQICSGPVVPSLRPNAVKELVENSLDAGATNVDLKLKDYGVDLIEVSGNGCGVEEENFEGFTLKHHTCKIQEFADLTQVETFGFRGEALSSLCALSDVTISTCRVSAKVGTRLVFDHYGKIIQKTPYPRPRGMTVSVKQLFSTLPVHHKEFQRNIKKKRACFPFAFCRDCQFPEASPAMLPVQPVELTPRSTPPHPCSLEDNVITVFSSVKNGPGSSR HPMSR3 (humancDNA) ACCESSION U38979 (SEQ ID NO: 13) 1 tttttagaaa ctgatgtttattttccatca accatttttc catgctgctt aagagaatat 61 gcaagaacag cttaagaccagtcagtggtt gctcctaccc attcagtggc ctgagcagtg 121 gggagctgca gaccagtcttccgtggcagg ctgagcgctc cagtcttcag tagggaattg 181 ctgaataggc acagagggcacctgtacacc ttcagaccag tctgcaacct caggctgagt 241 agcagtgaac tcaggagcgggagcagtcca ttcaccctga aattcctcct tggtcactgc 301 cttctcagca gcagcctgctcttctttttc aatctcttca ggatctctgt agaagtacag 361 atcaggcatg acctcccatgggtgttcacg ggaaatggtg ccacgcatgc gcagaacttc 421 ccgagccagc atccaccacattaaacccac tgagtgagct cccttgttgt tgcatgggat 481 ggcaatgtcc acatagcgcagaggagaatc tgtgttacac agcgcaatgg taggtaggtt 541 aacataagat gcctccgtgagaggcgaagg ggcggcggga cccgggcctg gcccgtatgt 601 gtccttggcg gcctagactaggccgtcgct gtatggtgag ccccagggag gcggatctgg 661 gcccccagaa ggacacccgcctggatttgc cccgtagccc ggcccgggcc cctcgggagc 721 agaacagcct tggtgaggtggacaggaggg gacctcgcga gcagacgcgc gcgccagcga 781 cagcagcccc gccccggcctctcgggagcc ggggggcaga ggctgcggag ccccaggagg 841 gtctatcagc cacagtctctgcatgtttcc aagagcaaca ggaaatgaac acattgcagg 901 ggccagtgtc attcaaagatgtggctgtgg atttcaccca ggaggagtgg cggcaactgg 961 accctgatga gaagatagcatacggggatg tgatgttgga gaactacagc catctagttt 1021 ctgtggggta tgattatcaccaagccaaac atcatcatgg agtggaggtg aaggaagtgg 1081 agcagggaga ggagccgtggataatggaag gtgaatttcc atgtcaacat agtccagaac 1141 ctgctaaggc catcaaacctattgatcgga agtcagtcca tcagatttgc tctgggccag 1201 tggtactgag tctaagcactgcagtgaagg agttagtaga aaacagtctg gatgctggtg 1261 ccactaatat tgatctaaagcttaaggact atggagtgga tctcattgaa gtttcagaca 1321 atggatgtgg ggtagaagaagaaaactttg aaggcttaat ctctttcagc tctgaaacat 1381 cacacatgta agattcaagagtttgccgac ctaactgaag ttgaaacttt cggttttcag 1441 ggggaagctc tgagctcactgtgtgcactg agcgatgtca ccatttctac ctgccacgcg 1501 ttggtgaagg ttgggactcgactggtgttt gatcacgatg ggaaaatcat ccaggaaacc 1561 ccctaccccc accccagagggaccacagtc agcgtgaagc agttattttc tacgctacct 1621 gtgcgccata aggaatttcaaaggaatatt aagaagacgt gcctgcttcc ccttcgcctt 1681 ctgccgtgat tgtcagtttcctgaggcctc cccagccatg cttcctgtac agcctgcaga 1741 actgtgagtc aattaaacctcttttcttca taaattaaaa aaaaa hPMSR3 (human protein) ACCESSION U38979 (SEQID NO: 24) MCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNSLGEVDRRGPREQTRAPATAAPDRPLGSRGAEAAEPQEGLSATVSACFQEQQENNTLQGPVSFKDVAVDFTQEEWRQLDPDSKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKEVEQGEEPWIMEGEFPCQHSPEPAKAIKPIDRKSVHQICSGPVVLSLSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM” hPMSL9 (human cDNA)ACCESSION NM_005395 (SEQ ID NO: 14) 1 atgtgtcctt ggcggcctag actaggccgtcgctgtatgg tgagccccag ggaggcggat 61 ctgggccccc agaaggacac ccgcctggatttgccccgta gcccggcccg ggcccctcgg 121 gagcagaaca gccttggtga ggtggacaggaggggacctc gcgagcagac gcgcgcgcca 181 gcgacagcag ccccgccccg gcctctcgggagccgggggg cagaggctgc ggagccccag 241 gagggtctat cagccacagt ctctgcatgtttccaagagc aacaggaaat gaacacattg 301 caggggccag tgtcattcaa agatgtggctgtggatttca cccaggagga gtggcggcaa 361 ctggaccctg atgagaagat agcatacggggatgtgatgt tggagaacta cagccatcta 421 gtttctgtgg ggtatgatta tcaccaagccaaacatcatc atggagtgga ggtgaaggaa 481 gtggagcagg gagaggagcc gtggataatggaaggtgaat ttccatgtca acatagtcca 541 gaacctgcta aggccatcaa acctattgatcggaagtcag tccatcagat ttgctctggg 601 ccagtggtac tgagtctaag cactgcagtgaaggagttag tagaaaacag tctggatgct 661 ggtgccacta atattgatct aaagcttaaggactatggag tggatctcat tgaagtttca 721 gacaatggat gtggggtaga agaagaaaactttgaaggct taatctcttt cagctctgaa 781 acatcacaca tgtaa hPMSL9 (humanprotein) ACCESSION NM_005395 (SEQ ID NO: 25)MCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNSLGEVDRRGPREQTRAPATAAPPRPLGSRGAEAAEPQEGLSATVSACFQEQQEMNTLQGPVSFKDVAVDFTQEEWRQLDPDEKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKEVEQGEEPWIMEGEFPCQHSPEPAKAINPIDRKSVHQICSGPVVLSLSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM”REFERENCES

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1. A method for making a hypermutable bacterium comprising the steps of:introducing into a bacterium a polynucleotide encoding a dominantnegative PMS2 mismatch repair protein, wherein the dominant negativePMS2 protein consists of the first 133 amino acids of PMS2, under thecontrol of an inducible transcription regulatory sequence; and inducingsaid inducible transcription regulatory sequence in said bacterium;whereby said bacterium becomes hypermutable.
 2. The method of claim 1wherein the dominant negative PMS2 mismatch repair protein is a dominantnegative human PMS2 protein consisting of the first 133 amino acids ofhuman PMS2.
 3. The method of claim 1 wherein the dominant negative PMS2mismatch repair protein is a dominant negative plant PMS2 proteinconsisting of the first 133 amino acids of a plant PMS2.
 4. The methodof claim 3 wherein said polynucleotide encoding a dominant negative PMS2mismatch repair protein comprises a truncation mutation at codon
 134. 5.The method of claim 2 wherein said polynucleotide encoding a dominantnegative PMS2 mismatch repair protein comprises a truncation mutation atcodon
 134. 6. A homogeneous composition of induced, cultured,hypermutable bacteria which comprise a polynucleotide encoding adominant negative mismatch repair protein under the control of aninducible transcription regulatory sequence, wherein said dominantnegative mismatch repair protein consists of the first 133 amino acidsof PMS2, wherein said dominant negative mismatch repair protein exerts adominant negative effect when expressed in said bacteria.
 7. Thehomogeneous composition of claim 6 wherein the dominant negativemismatch repair protein consists of the first 133 amino acids of humanPMS2.
 8. The method of claim 1 wherein the polynucleotide encoding adominant negative PMS2 mismatch repair protein comprises a truncationmutation at codon
 134. 9. A method for making a hypermutable bacteriumcomprising the steps of: introducing into a bacterium a polynucleotideencoding a dominant negative mismatch repair protein under the controlof an inducible transcription regulatory sequence, wherein said dominantnegative mismatch repair protein is a dominant negative PMSR3 mismatchrepair protein; and inducing said bacterium; wherein said dominantnegative PMSR3 mismatch repair protein exerts a dominant negative effecton mismatch repair when expressed in said bacterium, whereby saidbacterium becomes hypermutable.
 10. A homogeneous composition ofinduced, cultured, hypermutable bacteria which comprise a polynucleotideencoding a dominant negative PMSR3 mismatch repair protein under thecontrol of an inducible transcription regulatory sequence, wherein saiddominant negative PMSR3 mismatch repair protein exerts a dominantnegative effect when expressed in said bacteria.
 11. The composition ofclaim 6 wherein the dominant negative mismatch repair protein consistsof the first 133 amino acids of plant PMS2.
 12. The composition of 11wherein the dominant negative plant mismatch repair protein is anArabidopsis thaliana mismatch repair protein consisting of the first 133amino acids of Arabidopsis thaliana PMS2.
 13. The method of claim 3wherein the dominant negative plant PMS2 mismatch repair proteinconsists of the first 133 amino acids of Arabidopsis thaliana PMS2. 14.The method of claim 4 wherein the dominant negative plant PMS2 mismatchrepair protein consists of the first 133 amino acids of Arabidopsisthaliana PMS2.
 15. The homogeneous composition of claim 6 wherein thedominant negative mismatch repair protein is a dominant negativeArabidopsis thaliana mismatch repair protein which consists of the first133 amino acids of Arabidopsis thaliana PMS2.